Method to assay coenzyme Q10 in blood plasma or blood serum

ABSTRACT

A method is described for determining CoQ 10  concentrations in plasma samples. CoQ 10  in the plasma sample is oxidized by treating the sample with an oxidizing agent having a redox potential higher than the redox potential of CoQ 10 , such as, for example, para-benzoquinone. Following oxidation of the CoQ 10 , the CoQ 10  in the plasma sample is extracted with an alcohol, such as, for example, 1-propanol. The alcohol extract is analyzed using direct injection into the HPLC apparatus. This method achieves a rapid, accurate analysis of plasma CoQ 10  levels, which can be used for monitoring the bioavailability of orally administered CoQ 10  used as a food supplement or as an adjunctive therapy.

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/382,943 filed on May 23, 2002.

BACKGROUND OF THE INVENTION

[0002] Coenzyme Q₁₀ (2,3 dimethyl-5-methyl-6-decaprenyl benzoquinone)(“CoQ₁₀”) levels in whole blood and plasma have been the subject of muchinquiry as described, for example, in Tomasetti, M., Alleva, R.,Solenghi, M. D., Littarru, G. P., Distribution of antioxidants amongblood components and lipoproteins: significance of lipids/CoQ₁₀ ratio asa possible marker of increased risk for atherosclerosis. BioFactors, 9,231-240 (1999), the entire content of which is incorporated herein byreference. It is likely that plasma concentrations of CoQ₁₀ reflect anoverall metabolic demand, as discussed in Littarru, G. P., Lippa, S.,Oradei, A., Fiorini, R. M., Mazzanti, L., Metabolic and diagnosticimplications of human blood CoQ₁₀ levels, in Biomedical and ClinicalAspects of Coenzyme Q vol. VI, (eds. K. Folkers, G. P. Littarru, T.Yamagami), Elsevier North Holland, pp. 167-178 (1991), the entirecontent of which is incorporated herein by reference. In addition,together with other lipophilic antioxidants, CoQ₁₀ plays an intrinsicrole in protecting circulating lipoproteins against oxidative damage.Therefore, the concentration of CoQ₁₀ in lipoproteins and blood plasmacould be of clinical importance regarding oxidative stress andantioxidant defense. Increased levels of CoQ₁₀ enhance its antioxidantprotection, even though the potential to act as an antioxidant in vivoprobably depends not only on total CoQ₁₀ concentration, but also on itsredox status. The content of CoQ₁₀ in single classes of lipoproteins hasbeen found to be strictly correlated with CoQ₁₀ plasma concentration.Previous reports have shown that the LDL-cholesterol/CoQ₁₀ ratiosignificantly correlates with the total-cholesterol/HDL-cholesterolratio which is usually considered a risk factor for atherosclerosis asdescribed, for example in Alleva, R., Tomasetti, M., Bompadre, S.,Littarru, G. P., Oxidation of LDL and their subfractions: kineticaspects and CoQ₁₀ content. Molec Asp Med, 18, s105-s112 (1997), theentire content of which is incorporated herein by reference. Someeffective hypocholesterolemic agents, namely the statins, also lowerplasma CoQ₁₀ concentrations, owing to the common biosynthetic pathway ofcholesterol and the isoprenoide side chain of coenzyme Q as described,for example, in Mortensen, S. A., Leth, A., Agner, E., Rohde, M.,Dose-related decrease of serum coenzyme Q₁₀ during treatment withHMG-CoA reductase inhibitors. Molec Asp Med, 18, s137-s144 (1997), theentire content of which is incorporated herein by reference. Therefore,it would be desirable to have an effective, reliable, fast method tomeasure CoQ₁₀ concentrations in blood plasma or blood serum to monitorthe CoQ₁₀ levels in patients receiving hypocholesterolemic agents.

[0003] CoQ₁₀ is used as a food supplement or as an adjunctive therapy inseveral diseases and the blood plasma or blood serum levels achievedupon oral administration of CoQ10 can correlate with clinical efficacy.Tests of blood plasma or blood serum levels of CoQ₁₀ are useful formonitoring the bioavailability of orally administered coenzyme Q₁₀.

[0004] Several methods have been described for assaying either totalCoQ₁₀ or the reduced (ubiquinol-10, CoQ₁₀H₂) and oxidized(ubiquinone-10) forms in blood plasma, and several of these methods aredescribed in the following references, the entire contents of each ofwhich are incorporated herein by reference: Lang, J. K, Packer, L.,Quantitative determination of vitamin E and oxidized and reducedcoenzyme Q by high-performance liquid chromatography with in-lineultraviolet and electrochemical detection, J Chromatogr, 385, 109-117(1987); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski,M., Kohlschutter, A., High-performance liquid chromatography-coulometricelectrochemical detection of ubiquinol 10, ubiquinone 10, carotenoidsand tocopherols in neonatal plasma, in Methods in Enzymology, Vol 299,(Lester Packer Ed.), pp. 341-348, Academic Press, San Diego (1999);Podda, M., Weber, C., Traber, M. G., Milbradt, R., Packer, L., Sensitivehigh-performance liquid chromatography techniques for simultaneousdetermination of tocopherols, tocotrienols, ubiquinols and ubiquinonesin biological samples in Methods in Enzymology, Vol 299, (Lester PackerEd.), pp. 330-341, Academic Press, San Diego (1999); Finckh, B.,Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A.Monitoring of ubiquinol 10, ubiquinone 10, carotenoids and tocopherolsin neonatal plasma microsamples using high-performance liquidchromatography with coulometric electrochemical detection. Anal.Biochem, 232, 210-216 (1985); Okamoto, T., Fukunaga, Y., Ida, Y., Kishi,T., Determination of reduced and total ubiquinones in biologicalmaterials by liquid chromatography with electrochemical detection, J.Chromatogr, 430, 11-19 (1988); Grossi, G., Bargossi, A. M., Fiorella, P.L., Piazzi, S. Improved high-performance liquid chromatographic methodfor the determination of coenzyme Q₁₀ in plasma. J. Chromatogr., 593,217-226 (1988); Edlund, P. O., Determination of coenzyme CoQ₁₀,α-tocopherol and cholesterol in biological samples by coupled-columnliquid chromatography with coulometric and ultraviolet detection. J.Chromatogr, 425, 87-97 (1988); Lagendijk, J., Ubbink, J. B., Vermaak, W.J., Measurement of the ratio between the reduced and oxidized forms ofcoenzyme Q10 in human plasma as a possible marker of oxidative stress,J. Lipid Res, 37, 67-75 (1996); Yamashita, S., Yamamoto, Y.,Simultaneous detection of ubiquinol and ubiquinone in human plasma as amarker of oxidative stress, Anal. Biochem, 250, 66-73 (1997). As usedherein, ubiquinol means reduced CoQ₁₀ and ubiquinone means oxidizedCoQ₁₀.

[0005] The previous methods of analyzing blood plasma to assay theconcentration of CoQ₁₀ have several disadvantages. Prior methods requirethat the CoQ₁₀ be extracted from the plasma, followed by drying, whichconcentrates the extract. Losses in CoQ₁₀ can occur during the dryingand concentration step. In addition, these methods analyze the oxidizedform of CoQ₁₀, while most of the CoQ₁₀ in the plasma is in the reducedform. These methods rely on oxidation of the CoQ₁₀ during the extractionprocedure. UV methods for assaying CoQ₁₀ usually quantify the oxidizedcoenzyme at 275 nm. It is commonly assumed that CoQ₁₀ is completelyoxidized during the extraction and HPLC procedure, but this is notnecessarily the case when the sample is fresh and the reduced form ofCoQ₁₀ largely predominates. Accordingly, these methods can result inunderestimates of CoQ₁₀ concentration if all of the CoQ₁₀ is notoxidized.

[0006] The present invention is directed to a new, simplified method forevaluating total CoQ₁₀ in blood plasma or blood serum. The method of thepresent invention results in reduced time and cost for the analysis ofCoQ₁₀ in plasma or serum as compared to prior methods, and provides moreaccurate results.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1A shows a typical chromatogram of a plasma sample, beforeand after spiking with a known amount of standard. A typicalchromatogram of the standard alone is also shown.

[0008]FIG. 1B shows a diode array analysis of the peak of a standard ofCoQ₁₀ (50 ng).

[0009]FIG. 1C shows a diode array analysis of the peak obtained on aplasma sample analyzed by the method of the present invention.

[0010]FIG. 2 shows a linear correlation between the results obtained byanalysis of a sample for CoQ₁₀ using a reference electrochemicaldetection method and the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a method to assay theconcentration of CoQ₁₀ in blood plasma or blood serum. The sample ofblood plasma or blood serum is treated with an oxidizing agent having aredox potential higher than the redox potential of CoQ₁₀, followed byextraction with an alcohol and direct injection of the alcohol extractinto a High Performance Liquid Chromatography (“HPLC”) apparatus.

[0012] In a preferred embodiment of the method, the CoQ₁₀ in the plasmaor serum sample is oxidized by adding para-benzoquinone as an oxidizingagent. Extraction is performed using an alcohol, preferably n-propanol.The extract is then assayed by direct injection of the propanol extractinto the HPLC apparatus without bringing the extract to dryness. Themethod can be conducted on fresh plasma or serum samples since CoQ₁₀present in the sample is oxidized prior to propanol extraction.

[0013] The method may be performed using an oxidizing agent other thanbenzoquinone. For example, other oxidizing agents having a redoxpotential higher than the ubiquinone/ubiquinol couple may be used in themethod. Also, the extraction of the oxidized CoQ₁₀ in the plasma orserum sample may be performed using any appropriate alcohol known tothose skilled in the art. For example, 1-propanol, butanol or pentanolmay be used for the extraction of the oxidized CoQ₁₀.

[0014] A particularly preferred embodiment of the present method is setforth in the Example below. It should be understood that the descriptionset forth below is not intended to limit the invention in any way, andthose skilled in the art will readily understand that modifications tothe reagents, equipment or other parameters set forth below can be madewithout departing from the spirit or scope of the invention. Resultsobtained using the method on various samples are also described below.

EXAMPLE

[0015] Materials and Methods

[0016] Reagents. R.S. type methanol and n-propanol were used (obtainedfrom Carlo Erba, Rodano, Milan, Italy). Ethanol was R.S. plus grade(obtained from Carlo Erba, Rodano, Milan, Italy). Benzoquinone wasobtained from Sigma (St Louis, Mo., USA). Lithium perchlorate wasobtained from Aldrich (Steinheim, Germany).

[0017] Solutions for the ECD (electrochemical detection) chosen as areference method were filtered through a Nylon 66 membrane, 0.2 μm×47 mm(Supelco, Bellafonte, Pa., USA) and degassed. Pure Coenzyme Q₁₀ standardwas obtained from Kaneka (Osaka, Japan). Standard solutions were inethanol.

[0018] Samples. Blood was drawn from the cubital vein of laboratorystaff, after informed consent, and anti-coagulated with lithium heparin.Plasma obtained after centrifugation at 4,000 g for 15 min., at 4° C.was used fresh, or after storage at −80° C. for the day-to-day precisionassay.

[0019] In order to check the stability of total CoQ₁₀ in plasma,aliquots of 3 different samples were kept for 3 days at 4° C., roomtemperature (22° C.) and at −20° C. respectively.

[0020] High Performance Liquid Chromatography. The HPLC apparatusconsisted of a Beckman System pump model 126, a detector model 166(Beckman Instruments, San Ramon, Calif., USA) and an injector equippedwith a 2001 μl loop (Rheodyne 7725i obtained from Supelco, Milano,Italy). The column was a Supelcosil LC 18 (obtained from Supelco,Milano, Italy) 25 cm×0.46 cm i.d. 5μ, precolumn LC 18S, 2 cm. (obtainedfrom Supelco, Milano, Italy). An in line filter A-701 (obtained fromUpchurch Scientific, Inc., Oak Harbor, Wash., USA) was placed betweenthe injector and the precolumn. The photodiode array detector for the UVspectrum analysis of the CoQ₁₀ peak was a SPD-M (Shimadzu, Tokyo).

[0021] Description of the Analysis. 200 μl of a blood plasma sample weresupplemented with 50 μl of a 1,4 benzoquinone solution (2 mg/ml) in atest tube and vortexed for 10 seconds. After 10 minutes, 1 ml ofn-propanol was added. The test tube was vortexed for 10 seconds andcentrifuged at 10,000 rpm for 2 minutes in order to spin down theprotein precipitate. 200 μl of the supernatant were injected into theHPLC. The supernatant, placed in a capped test tube, was stable for upto three days when kept at 22° C. Mobile phase was constituted byethanol-methanol (65%-35%) and the flux was 1 ml/min. UV detection wasperformed at 275 nm.

[0022] 200 μl of different concentrations of pure oxidized CoQ₁₀ wereinjected as standards. Working solutions of the standards were inpropanol:water (5:1), i.e. the same propanol:water ratio as for thesamples. Peak area analysis was performed by a Beckman Gold Data System(DOS version).

[0023] Coulometric analysis. Coulometric analysis of CoQ₁₀ was performedaccording to a standard procedure described in Alleva, R., Tomasetti,M., Bompadre, S., Littarru, G. P., Oxidation of LDL and theirsubfractions: kinetic aspects and CoQ₁₀ content. Molec Asp Med, 18,s105-s112 (1997). Ubiquinol/ubiquinone separation was performed on anODS reversed phase column (Supelcosil LC 18, 15×4.6 mm i.d. 3 μm,Supelco, Milano, Italy) using a mobile phase constituted by 50 mM sodiumperchlorate in methanol:ethanol (80:20), at flow rate of 1 ml/min. ACoulochem II, model 5200 electrochemical detector (ESA, Bedford, Mass.,USA), with the analytical cell set at −0.5 V and +0.35 V was used todetect the oxidized and reduced forms of CoQ₁₀.

[0024] Recovery, Accuracy and Precision. Recovery of CoQ₁₀ was based ona comparison between the peaks obtained by spiking samples withincreasing concentrations of oxidized CoQ₁₀ and the corresponding peaksof the standard. Recovery was documented at 3 concentrations (1.16,2.32, and 3.48 μM) with triplicate determinations for eachconcentration. Intra-assay accuracy and precision were determined using4 samples, the value of which had been certified by a reference ECDmethod; each level was assayed 5 times for the intra-assay accuracy andprecision test. Inter-assay accuracy and precision were determined overa two-month period using a quality control sample (n=21).

[0025] Results

[0026] Chromatography and recovery. Typical, representativechromatograms of both a standard and a plasma sample are shown in FIG.1A. Diode array analysis of the peak with the same retention time as theCoQ₁₀ standard is also shown in FIGS. 1B and 1C. Spiking of a samplecontaining an initial concentration of 0.29 μM with 1.16, 2.32 or 3.48μM CoQ₁₀ yield a recovery of 96.3, 98.1 and 98.5% as shown in Table 1.TABLE 1 Recovery of exogenous CoQ₁₀ added to a plasma sample Measured(pM) Recovery Basal concentration 0.29 Spiking with CoQ₁₀ 1.16 μM 1.4096.3% Spiking with CoQ₁₀ 2.32 μM 2.56 98.1% Spiking with CoQ₁₀ 3.48 μM3.72 98.5%

[0027] Calibration curves. Calibration curves constructed usingpropanol/water solutions of pure CoQ₁₀ as described in the Materials andMethods section above showed linearity over a concentration range of7.9-579 nM, corresponding to a concentration of 47.4-3474 nM CoQ₁₀ inplasma. Correlation coefficients (r²) for 20 calibration curves obtainedover a two month period ranged from 0.98 to 0.999 The limit ofquantitation was 0.037 μM (1.23 nmoles in column) with a precision of10.52%.

[0028] Accuracy and precision. Within run (intra-assay) precision (CV %)and accuracy, determined as deviation from nominal values, appear inTable 2. Four samples with different, decreasing concentrations of totalCoQ₁₀, having a nominal value previously determined by theelectrochemical detection method chosen as reference, were analysed fivetimes each. As shown in Table 2, CV % becomes consistently higher than 5only for samples less than 0.06 μM. Normal plasma values of CoQ₁₀typically range between 0.75 and 0.98 μM. Deviation from nominal valueswas never higher than 4.7%. TABLE 2 Within run accuracy and precisionExpected Measured concentration concentration (nM) (nM) Deviation % C.V.% 894 911 1.9% 1.6% 447 432 −3.4% 5.3% 178 170 −4.7% 5.7% 36 37 2.6%10.5%

[0029] Day-to-day precision, conducted over a two month period, issummarized in Table 3. For values corresponding to normal plasmaconcentrations, accuracy and precision were comparable to that obtainedin the intra-assay conditions. TABLE 3 Day to day accuracy and precisiontest Nominal concentration of sample 0.81 (μM) Mean of 21 measurements(μM) 0.80 % deviation from nominal value 0.7 C.V. % 2.16

[0030] Comparison with the ECD method. Ten samples were analysed, inparallel, both by the electrochemical method (ECD) and by the method ofthe present invention (UV), and the results are shown in Table 4. Thesesamples were chosen on the basis of their different levels of totalCoQ₁₀ and different extent of reduction. Correlation between the resultsof total CoQ₁₀ obtained by the two methods appears in FIG. 2. TABLE 4Results from a group of samples analysed by electrochemical (ECD) and byUV detection ECD Detection UV Detection Oxidized Reduced Total CoQ₁₀Total CoQ₁₀ Sample CoQ₁₀ (μM) CoQ₁₀ (μM) (μM) Red/Tot (%) (μM) %Variation A 0.95 1.62 2.57 63.09% 2.84 10.27% B 1.47 0.30 1.78 17.12%1.75 −1.44% C 0.02 0.74 0.76 97.36% 0.83 9.44% D 2.01 0.10 2.11 4.80%2.18 3.50% E 0.30 1.02 1.32 77.56% 1.22 −7.51% F 0.45 0.05 0.51 10.76%0.48 −6.62% G 2.11 0.40 2.51 15.92% 2.37 −5.53% H 0.15 1.69 1.84 92.03%2.00 8.89% I 0.58 0.49 1.07 45.91% 1.03 −3.38% L 0.94 0.84 1.78 47.03%1.79 0.71%

[0031] Stability. The results obtained on 3 different samples stored for1,2 or 3 days at different temperatures are shown in Table 5. TotalCoQ₁₀ was practically stable for at least three days even when kept atroom temperature. TABLE 5 Stability of CoQ₁₀ values at different storageconditions and times Time after blood withdrawal (hours) 1 h. 24 h.27/34 48 h. 72 h. Storage Mean St. Mean St. Mean St. Mean St. Sampleconditions μM Dev. μM Dev. μM Dev. μM Dev. A Control 1.73 ± 0.05 Room1.91 ± 0.03 1.81 ± 0.00 1.93 ± 0.03 Temp. 0-4° C. 1.97 ± 0.03 1.82 ±0.11 1.82 ± 0.16 −20° C. 1.84 ± 0.05 1.91 ± 0.08 1.97 ± 0.05 B Control1.06 ± 0.03 Room 1.11 ± 0.04 1.05 ± 0.02 1.02 ± 0.03 Temp. 0-4° C. 1.06± 0.05 1.03 ± 0.08 1.11 ± 0.03 −20° C. 1.14 ± 0.06 1.00 ± 0.00 1.09 ±0.03 C Control 2.68 ± 0.11 Room 2.72 ± 0.12 2.80 ± 0.11 2.83 ± 0.09Temp. 0-4° C. 2.81 ± 0.15 2.78 ± 0.01 2.94 ± 0.07 −20° C. 2.84 ± 0.112.80 ± 0.08 2.93 ± 0.08

[0032] Discussion of Results. Prior methods for evaluating CoQ₁₀ inplasma and biological tissues are based on alcohol-hexane extraction;the extract is brought to dryness and injected into the HPLC apparatus,where CoQ₁₀ is usually revealed and quantified at 275 nm or byelectrochemical detection. An internal standard, such as CoQ₈ or CoQ₉,is often added to the sample before extraction, in order to quantify arecovery. In all previously described methods, the organic solventextract is brought to dryness and therefore concentrated beforeinjection into the HPLC.

[0033] In the present method the sample is only diluted with propanoland then injected into the HPLC. Direct injection of the propanolextract also makes the procedure particularly simple and fast. Prioroxidation of reduced CoQ₁₀ with para-benzoquinone eliminates thepossibility of underestimating total CoQ₁₀ in fresh samples. In factfresh samples contain almost exclusively reduced CoQ₁₀ and the usualextraction procedures are often not sufficient to completely oxidizeubiquinol. On the other hand UV detection at 275 only revealsubiquinone. The chromatographic features and the diode array analysis ofthe peak show a selective separation of CoQ₁₀, with no superimposedpeaks. Reproducibility of the method is shown by a CV below 2% forsamples having normal values of plasma CoQ₁₀ and about 5% for sampleshaving a quarter of the normal amount.

[0034] The rather low retention time for CoQ₁₀, under thechromatographic conditions makes the analysis fast enough and the peakdoes not overlap with any other components, as shown by similar valuesobtained using the ECD method and by the diode array analysis of thepeak.

[0035] The efficiency of CoQ₁₀ extraction by the method of the presentinvention appears to be very satisfactory as the addition of 1, 2 or 3μg of CoQ₁₀ to a sample leads to a 96.3-98.5% quantitative recovery (seeTable 4) which makes the use of an internal standard unnecessary. Thisis in agreement with data from Edlund's work (Edlund, P. O.,Determination of coenzyme CoQ₁₀, α-tocopherol and cholesterol inbiological samples by coupled-column liquid chromatography withcoulometric and ultraviolet detection. J. Chromatogr, 425, 87-97 (1988))where it appears that a dilution of the sample with n-propanol (1:4)leads to a 100% recovery.

[0036] Correlation with the reference ECD method gave an r=0.98(p<0.0001) as shown in Table 4. The data of Table 4 also show theefficiency of the method in properly quantifying differentconcentrations of CoQ₁₀ and different extents of reduction. Freshsamples have their CoQ₁₀ almost completely reduced, see Lagendijk, J.,Ubbink, J. B., Vermaak, W. J., Measurement of the ratio between thereduced and oxidized forms of coenzyme Q10 in human plasma as a possiblemarker of oxidative stress, J. Lipid Res, 37, 67-75 (1996); Yamashita,S., Yamamoto, Y., Simultaneous detection of ubiquinol and ubiquinone inhuman plasma as a marker of oxidative stress, Anal. Biochem, 250, 66-73(1997). Therefore, the testing described above also included somesamples which had been frozen and thawed several times, where CoQ₁₀ waspartially or almost completely oxidised. It is evident that the extentof reduction of CoQ₁₀ in the sample does not affect the final result.

[0037] For practical reasons the propanol extract can be kept in therefrigerator up to 3 days before injection into the HPLC. Severalsamples were checked by analysing them 1 hour, 1, 2 and 4 days afterdrawing the blood, keeping the samples at −20° C., 0-4° C., or +22° C.(room temperature) and the results, as far as total CoQ₁₀ was concerned,were the same for the same sample whether kept at different temperaturesor for different lengths of time (presumably only the reduced/oxidisedCoQ₁₀ ratio changed). There was no need to add any stabilizing agent tothe sample.

[0038] The data reported above demonstrates that the method of thepresent invention is simple and reliable, it minimizes sample handling,allows a quantitative recovery of CoQ₁₀ and it does not need an internalstandard. Data not reported show that it is adaptable to semi-automatedprocedures and can be applied to tissue homogenates.

[0039] As will be recognized by those of ordinary skill in the art basedon the teachings herein, numerous changes and modifications may be madeto the above-described methods without departing from its spirit orscope as defined in the appended claims. Accordingly, this detaileddescription of preferred embodiments is to be taken in an illustrative,as opposed to a limiting sense.

We claim:
 1. A method for determining the concentration of CoQ₁₀ inblood plasma or blood serum, comprising the steps of: (a) obtaining asample of blood plasma or blood serum from a human; (b) treating thesample with an oxidizing agent having a redox potential greater than theredox potential of CoQ₁₀; (c) extracting the oxidized CoQ₁₀ from thesample using an alcohol; and (d) analyzing the alcohol to determine theconcentration of CoQ₁₀.
 2. The method of claim 1, wherein the oxidizingagent having a redox potential greater than the redox potential of CoQ₁₀is 1,4 benzoquinone.
 3. The method of claim 2, wherein the alcohol isselected from the group consisting of n-propanol, butanol and pentanol4. The method of claim 2, wherein the alcohol is 1-propanol.
 5. Themethod of claim 1, wherein the step of analyzing the alcohol isperformed by High Performance Liquid Chromatography.
 6. A method fordetermining the concentration of CoQ₁₀ in blood plasma or blood serum,comprising the steps of: (a) obtaining a sample of blood plasma or bloodserum from a human; (b) depositing an aliquot of the sample in acontainer; (c) adding to the sample in the container a sufficient volumeof an oxidizing agent having a redox potential greater than the redoxpotential of CoQ₁₀ to oxidize CoQ₁₀ in the sample; (d) vortexing thesample containing the oxidizing agent; (e) allowing the vortexed samplecontaining the oxidizing agent to stand for a sufficient period of timefor the oxidizing agent to oxidize CoQ₁₀ contained in the sample; (f)adding an alcohol to the sample containing the oxidizing agent toextract the oxidized CoQ₁₀ from the sample; (g) vortexing the samplecontaining the alcohol; (h) centrifuging the sample containing thealcohol to spin down the protein precipitate; (i) removing an aliquot ofthe supernatant from the centrifuged sample and analyzing the aliquot ofsupernatant to determine the concentration of CoQ₁₀ in the sample. 7.The method of claim 6, wherein the oxidizing agent is 1,4 benzoquinone.8. The method of claim 7, wherein the alcohol is selected from the groupconsisting of n-propanol, butanol and pentanol.
 9. The method of claim7, wherein the alcohol is 1-propanol.
 10. The method of claim 6, whereinthe aliquot of supernatant is analyzed using High Performance LiquidChromatography.
 11. A method for determining the concentration of CoQ₁₀in blood plasma or blood serum, comprising the steps of: (a) obtaining asample of blood plasma or blood serum from a human and depositing 200 μlof the sample in a test tube; (b) adding 50 μl of 1,4-benzoquinonesolution to the sample contained in the test tube, wherein the1,4-benzoquinone solution contains 2 mg 1,4-benzoquinone/ml solution,and vortexing the sample and 1,4-benzoquinone for about 10 seconds; (c)allowing the test tube containing the vortexed sample to stand for about10 minutes; (d) adding 1 ml of 1-propanol to the test tube and vortexingthe resulting the sample and the alcohol in the test tube for about 10seconds; (e) centrifuging the test tube at about 10,000 rpm for about 2minutes; (f) removing 200 μl of supernatant from the test tube andinjecting the supernatant into a High Performance Liquid Chromatographyapparatus to determine the the concentration of CoQ₁₀ in the sample.